WO2018232467A1 - Thérapie par lymphocytes t adoptive 2 - Google Patents

Thérapie par lymphocytes t adoptive 2 Download PDF

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Publication number
WO2018232467A1
WO2018232467A1 PCT/AU2018/050630 AU2018050630W WO2018232467A1 WO 2018232467 A1 WO2018232467 A1 WO 2018232467A1 AU 2018050630 W AU2018050630 W AU 2018050630W WO 2018232467 A1 WO2018232467 A1 WO 2018232467A1
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Prior art keywords
cells
antigen
population
hla
composition
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PCT/AU2018/050630
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English (en)
Inventor
Emily BLYTH
Leighton CLANCY
David Gottlieb
Kenneth MICKLETHWAITE
Original Assignee
The Westmead Institute for Medical Research
The University Of Sydney
Western Sydney Local Health District
New South Wales Health Pathology
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Priority claimed from AU2017902407A external-priority patent/AU2017902407A0/en
Application filed by The Westmead Institute for Medical Research, The University Of Sydney, Western Sydney Local Health District, New South Wales Health Pathology filed Critical The Westmead Institute for Medical Research
Priority to US16/625,443 priority Critical patent/US11951127B2/en
Priority to EP18820696.5A priority patent/EP3641807A4/fr
Priority to AU2018288386A priority patent/AU2018288386A1/en
Publication of WO2018232467A1 publication Critical patent/WO2018232467A1/fr
Priority to AU2021286255A priority patent/AU2021286255A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A61K39/001153—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4641—Fungal antigens, e.g. Trichophyton, Aspergillus or Candida
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464452—Transcription factors, e.g. SOX or c-MYC
    • A61K39/464453—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates to T cell products with activity against a fungal antigen, a viral antigen or a tumour antigen, wherein the product comprises a defined number or defined ratio of T cells.
  • Invasive pathogenic infections are significant causes of morbidity and mortality, particularly in patients undergoing haematological stem cell transplantation who are increasingly susceptible to infection.
  • T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity and, to a lesser degree the adaptive immune response. Generally, T cells are distinguished from other lymphocytes (e.g., B cells and natural killer cells) by the presence of T cell receptors (TCRs). T cells have diverse roles, which are accomplished by differentiation of distinct populations of T cells, recognizable by discrete gene expression profiles and phenotypes.
  • T cell populations include naive T cells, T helper cells (T H cells), terminally differentiated effector T cells (T eff cells), effector memory T cells (T em cells), central memory T cells (T cm cells), stem cell memory cells (Tscm), cytotoxic T cells (CTLs) and regulatory T cells (Treg cells).
  • T H cells T helper cells
  • T eff cells terminally differentiated effector T cells
  • T em cells effector memory T cells
  • T cm cells central memory T cells
  • Tscm stem cell memory cells
  • CTLs cytotoxic T cells
  • Reg cells regulatory T cells
  • Fungus, viral and tumour-specific T cell production is fundamental to the body's defence against invasive diseases, and defective in vitro T cell immunity has been shown to correlate with the period when patients are at highest risk of infection.
  • the present disclosure is based on the inventors' identification of a defined T cell product with specificity against a fungal, viral or tumour pathogen.
  • the inventors isolated T cells from peripheral blood and hematopoietic stem cells mobilized with G-CSF and expanded pathogen (i.e., fungal, viral or tumour) specific T cells using fungal antigens from fungal extracts, viral antigens from viruses or tumour antigens.
  • pathogen i.e., fungal, viral or tumour
  • the inventors prepared compositions comprising a defined number or defined ratio of at least two populations of T cells where each population of T cells is reactive with a fungal antigen, a viral antigen or a tumour antigen.
  • Previous multipathogen T cell compositions were undefined in their content of T cells specific for each pathogen and, as a consequence, of unknown specificity. By preparing compositions based on defined T cell populations, the inventors of the present disclosure proceeded against this conventional wisdom, and as such prepared compositions with broader protection.
  • the present disclosure is broadly directed to a composition comprising at least two populations of T cells each population reactive with a fungal antigen, a viral antigen or a tumour antigen, and wherein the composition comprises a defined number or defined ratio of T cells in each population.
  • the T cells are isolated from a sample from a subject, e.g. a blood sample or fraction thereof (e.g., buffy coat fraction or peripheral blood mononuclear cell fraction) or a thymus or part thereof.
  • a sample e.g. a blood sample or fraction thereof (e.g., buffy coat fraction or peripheral blood mononuclear cell fraction) or a thymus or part thereof.
  • the present disclosure also encompasses a method additionally comprising providing or obtaining a sample from a subject.
  • a sample may have been isolated previously from a subject, e.g., the method is performed in vitro or ex vivo.
  • the population of cells can also be an isolated population of cells, e.g., produced using tissue culture techniques.
  • each population of T cells are expanded, e.g., in vitro or ex vivo.
  • the isolated cells are culture expanded.
  • the isolated and/or expanded cell population of the present disclosure is not necessarily the same as naturally-occurring T cell populations. This is because the T cells in the population are expanded against a fungal, a viral or a tumour antigen and multiple T cell populations are combined in a way which does not necessarily occur in nature. Moreover, each population of T cells are expanded against individual antigens and the composition prepared from a combination of at least two T cell populations that may not occur in a subject at the same time. Furthermore, each population of T cells are expanded against individual antigens (i.e., a subset of infective organisms) as opposed to all antigens that may occur in a subject at the same time.
  • the fungal antigen is in the form of a fungal extract.
  • the fungal extract is a water soluble fungal lysate.
  • the water soluble lysate is formed from cellular extracts.
  • the water soluble lysate is formed from lysed fungal spores.
  • the water soluble lysates are produced by lysing germinated spores of the fungus in water and obtaining the lysate.
  • the water soluble lysates are obtained by homogenizing germinated spores of the fungus in water and obtaining the lysate.
  • the viral antigen is in the form of overlapping peptides from viral proteins or a lysate of virally infected cells.
  • the viral antigen is a viral protein or a peptide or peptides from a viral protein or is within a lysate of a virus infected cell or is expressed by a cell modified to express the viral antigen.
  • the tumour antigen is in the form of overlapping peptides from tumour proteins.
  • the tumour antigen is a tumour protein or a peptide or peptides from a tumour protein or is expressed by a cell modified to express the tumour antigen.
  • Exemplary T cell populations include naive T cells, T helper cells (3 ⁇ 4 cells), terminally differentiated effector T cells (T eff cells), effector memory T cells (T em cells), central memory T cells (T cm cells), cytotoxic T cells (CTLs) and regulatory T cells (Treg cells).
  • T helper cells (3 ⁇ 4 cells)
  • T eff cells terminally differentiated effector T cells
  • T em cells effector memory T cells
  • T cm cells central memory T cells
  • CTLs cytotoxic T cells
  • Treg cells regulatory T cells
  • the present disclosure provides a composition comprising at least two populations of T cells.
  • the composition comprises two populations of T cells, or three populations of T cells, or four populations of T cells, or five populations of T cells, or six populations of T cells, or seven populations of T cells, or eight populations of T cells, or nine populations of T cells, or ten populations of T cells, or eleven populations of T cells, or twelve populations of T cells, or thirteen populations of T cells, or fourteen populations of T cells, or fifteen populations of T cells, or sixteen populations of T cells, or seventeen populations of T cells, or eighteen populations of T cells, or nineteen populations of T cells, or twenty populations of T cells.
  • the composition comprises four populations of T cells.
  • the present disclosure provides a composition comprising at least two T cell populations wherein each population comprises a defined number or defined ratio of T cells.
  • the defined number or defined ratio of T cells in each population is based on a) the total number of nucleated cells; b) the number of CD3+ cells, or CD4+ cells, or CD8+ cells; c) the number of activation marker positive cells; and/or the number of cytokine producing cells on re-stimulation.
  • the defined number or defined ratio of T cells in each population is based on the total number of nucleated cells.
  • the defined number or defined ratio of T cells in each population is based on the number of CD3+ cells, or CD4+ cells, or CD8+ cells.
  • the defined number or defined ratio of T cells in each population is based on the number of activation marker positive cells.
  • the composition comprises an equal number of T cells in each population.
  • the composition comprises at least two populations of T cells, wherein each population is combined in a ratio of between about 1 : 1 and about 1 :99.
  • each population of T cells is combined in a ratio of about 1 :1, or about 1 :2, or about 1 : 3, or about 1 :4, or about 1:5, or about 1 :6, or about 1 :7, or about 1 :8, or about 1 :9, or about 1 :10.
  • each population of T cells is combined in a ratio of about 1 : 10, or about 1 :15, or about 1 :20, or about 1 :25, or about 1 :30, or about 1 :35, or about 1:40, or about 1 :45, or about 1:50, or about 1 :55, or about 1 :60, or about 1 :65, or about 1 :70, or about 1 :75, or about 1:80, or about 1 :85, or about 1 :90, or about 1 :95, or about 1 :99.
  • the defined number or defined ratio is based on a subject's clinical diagnosis.
  • the defined number or defined ratio is based on a subject's serology or infectious status.
  • the defined number or defined ratio is based on a subject's previous serology or infectious status.
  • the defined number or defined ratio is based on a known profile for an infection.
  • the composition comprises a total of between about lxl0 7 cells/m 2 and about 10xl0 7 cells/m 2 .
  • the composition comprises a
  • the composition comprises a total of about 4xl0 7 cells/m 2 .
  • each population of T cells in the composition comprises between about lxl0 6 cells/m 2 and about 10xl0 7 cells/m 2 of total nucleated cells. In one example, each population of T cells in the composition comprises between about lxl0 6 cells/m 2 and about 10xl0 6 cells/m 2 of total nucleated cells. For example, each
  • * 6 2 population of T cells in the composition comprises a total of about 1x10 cells/m , or
  • the composition comprises a total of about 3.5x10 6 of total nucleated cells/m 2 .
  • each population of T cells in the composition comprises between about lxl0 7 cells/m 2 and about 10xl0 7 cells/m 2 of total nucleated cells.
  • each population of T cells in the composition comprises a total of about 1x10 cells/m , or about 1.5x10 cells/m , or about 2x10 cells/m , or about
  • 2.5x10 cells/m or about 3x10 cells/m , or about 3.5x10 cells/m , or about 4x10 cells/m , or about 4.5x10 cells/m , or about 5x10 cells/m , or about
  • composition of the present disclosure comprises about 60-
  • the composition comprises about 70-80% CD3+ cells, such as about 70% CD3+ cells, or about 75% CD3+ cells, or about 80% CD3+ cells.
  • the composition comprises about 80-90%) CD3+ cells, such as about 85% CD3+ cells, or about 90% CD3+ cells.
  • the composition comprises about 90-100% CD3+ cells, such as about 95% CD3+ cells, or about 100% CD3+ cells.
  • the percentage of CD3+ T cells is detected by flow cytometry analysis of cells expressing CD3.
  • the composition of the present disclosure comprises about 10- 40% CD4+ cells, or about 10-20%, or about CD4+ cells, or about 20-30% CD4+ cells, or about 3-40% CD4+ cells, or about 50-100% CD4+ cells, or about 60-100% CD4+ cells, or about 60-90% CD4+ cells.
  • the composition comprises about 60- 70% CD4+ cells, such as about 60% CD4+ cells, or about 65% CD4+ cells, or about 70% CD4+ cells.
  • the composition comprises about 70-80%o CD4+ cells, such as about 75% CD4+ cells, or about 80% CD4+ cells.
  • the composition comprises about 80-90%) CD4+ cells, such as about 85% CD4+ cells, or about 90% CD4+ cells.
  • the composition comprises about 90-100% CD4+ cells, such as about 95% CD4+ cells, or about 100% CD4+ cells.
  • the percentage of CD4+ T cells is detected by flow cytometry analysis for cells expressing CD4.
  • composition of the present disclosure comprises about 10-
  • the composition comprises about 60- 70% CD8+ cells, such as about 60% CD8+ cells, or about 65% CD8+ cells, or about 70%) CD8+ cells.
  • the composition comprises about 70-80% CD8+ cells, such as about 75% CD8+ cells, or about 80% CD8+ cells.
  • the composition comprises about 80-90%> CD8+ cells, such as about 85%> CD8+ cells, or about 90% CD8+ cells.
  • the composition comprises about 90-100% CD8+ cells, such as about 95% CD8+ cells, or about 100% CD8+ cells.
  • the percentage of CD8+ T cells is detected by flow cytometry analysis for cells expressing CD8.
  • composition comprising at least two T cell populations each population reactive with a fungal antigen, a viral antigen or a tumour antigen.
  • composition comprises:
  • T cells each population reactive with a viral antigen and one or more population(s) of T cells reactive with a tumour antigen; or g) one or more population(s) of T cells each population reactive with a fungal antigen, one or more population of T cells each population reactive with a viral antigen and one or more population(s) of T cells each population reactive with a tumour antigen.
  • the composition comprises at least two T cell populations, wherein each population is reactive with a fungal antigen.
  • the composition does not comprise a population of T cells reactive with a viral or tumour antigen.
  • the composition comprises at least two T cell populations, wherein each population is reactive with a viral antigen.
  • the composition does not comprise a population of T cells reactive with a fungal antigen or a tumour antigen.
  • the composition comprises at least two T cell populations, wherein each population is reactive with a tumour antigen.
  • the composition does not comprise a population of T cells reactive with a fungal antigen or a viral antigen.
  • the composition comprises at least two T cell populations wherein at least one population of T cells is reactive with a fungal antigen and at least one population of T cells is reactive with a viral antigen.
  • the composition does not comprise a population of T cells reactive with a tumour antigen.
  • the composition comprises at least two T cell populations wherein at least one population of T cells is reactive with a fungal antigen and at least one population of T cells is reactive with a tumour antigen.
  • the composition does not comprise a population of T cells reactive with a viral antigen.
  • the composition comprises at least two T cell populations wherein at least one population of T cells is reactive with a viral antigen and at least one population of T cells is reactive with a tumour antigen.
  • the composition does not comprise a population of T cells reactive with a fungal antigen.
  • the composition comprises at least two T cell populations wherein at least one population of T cells is reactive with a fungal antigen, at least one population of T cells is reactive with a viral antigen and at least one population of T cells is reactive with a tumour antigen.
  • the composition comprises a population of T cells reactive with a fungal antigen from a fungal extract selected from the group consisting of Aspergillus terreus, Candida krusei, Rhizopus oryzae, A. fumigatus, A. flavus, A. terreus, C. albicans, C. krusei, F. oxysporum, F. solani and L. prolificans.
  • the composition comprises a population of T ce Is reactive with an extract of C. krusei and a population of T cells reactive with an extract of A. terreus.
  • the composition comprises a population of T cells reactive with an extract of A. fumigatus.
  • the composition comprises a population of T eel Is reactive with an extract of C. krusei.
  • the composition comprises a population of T eel Is reactive with an extract of A. terreus.
  • the composition comprises a population of T eel Is reactive with an extract of R. oryzae
  • the composition comprises a population of T eel Is reactive with an extract of A. flavus.
  • the composition comprises a population of T eel Is reactive with an extract of C. albicans.
  • the composition comprises a population of T eel Is reactive with an extract of F. oxysporum.
  • the composition comprises a population of T eel Is reactive with an extract of F. solani.
  • the composition comprises a population of T eel Is reactive with an extract of L. prolificans.
  • the composition comprises a population of T eel Is reactive with a viral antigen from a virus selected from the group consisting of cytomegalovirus, Epstein-Barr virus, adenovirus, varicella zoster virus, influenza and BK virus, John Cunningham virus, respiratory syncytial virus, parainfluenzae, rhinovirus, human metapneumovirus, herpes simplex virus 1, herpes simplex virus II, human herpesvirus 6, human herpesvirus 8, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, rotavirus, papillomavirus, parvovirus and mixtures thereof.
  • a virus selected from the group consisting of cytomegalovirus, Epstein-Barr virus, adenovirus, varicella zoster virus, influenza and BK virus, John Cunningham virus, respiratory syncytial virus, parainfluenzae, rhinovirus, human metapneu
  • the composition comprises a population of T cells reactive with cytomegalovirus, a population of T cells reactive with Epstein-Barr virus and a population of T cells reactive with adenovirus.
  • the composition comprises a population of T cells reactive with cytomegalovirus, a population of T cells reactive with Epstein-Barr virus, a population of T cells reactive with adenovirus and a population of T cells reactive with varicella zoster virus.
  • the composition comprises a population of T cells reactive with cytomegalovirus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with CMV pp65.
  • the composition comprises a population of T cells reactive with Epstein-Barr virus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with EBV BZLF1, LMP2A and/or EBNA1.
  • the composition comprises a population of T cells reactive with adenovirus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with AdV hexon.
  • the composition comprises a population of T cells reactive with varicella zoster virus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with BK virus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with BKV VP1, VP2, STA and/or LTA.
  • the composition comprises a population of T cells reactive with the influenza virus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with the JC virus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with RSV or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with the parainfiuenzae virus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with the rhinovirus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with the human metapneumovirus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with HS V 1 or HS V II or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with the HFIV6 or HHV8 or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with hepatitis A virus or hepatitis B virus or hepatitis C virus or hepatitis E virus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with the rotavirus or a viral antigen thereof. In one example, the composition comprises a population of T cells reactive with a papillomavirus or a viral antigen thereof.
  • the composition comprises a population of T cells reactive with a parvovirus or a viral antigen thereof.
  • the present disclosure provides a composition comprising a population of T cells reactive with a tumour antigen selected from the group consisting of Wilms tumour antigen (WT1), beta chain of human chorionic gonadotropin (hCG beta) antigen, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), CD 19, CD20, CA-125, epithelial tumor antigen (ETA), abnormal products of ras, p53, glycosphingolipid GD2, prostatic acid phosphatase (PAP), preferentially expressed antigen in melanoma (PRAME), B melanoma antigen (BAGE), cancer-testis antigen (NY ESO-1), sarcoma antigen 1 (SAGE), helicase antigen (HAGE), cancer-germline antigen (GAGE), prostein (P501S), six-transmembrane epithelial antigen of the prostate (STEAP), Plu-1, human achaete-scute homolog-1 (hASH), WT
  • the composition comprises a population of T cells reactive with an extract of C. krusei and a population of T cells reactive with an extract of A. terreus.
  • the composition comprises a population of T cells reactive with a Wilms tumour antigen (WT1).
  • WT1 Wilms tumour antigen
  • the composition comprises a population of T cells reactive with an extract of A. fumigatus, a population of T cells reactive with cytomegalovirus, a population of T cells reactive with Epstein-Barr virus and a population of T cells reactive with adenovirus.
  • the composition comprises a population of T cells reactive with cytomegalovirus, a population of T cells reactive with Epstein-Barr virus, a population of T cells reactive with adenovirus and a population of T cells reactive with varicella zoster virus.
  • the composition additionally comprises a population of T cells reactive with WT1.
  • administration of the composition of the present disclosure to a subject confers a therapeutic or protective immune response.
  • the composition confers an immune response against a fungal pathogen or a viral pathogen or a tumour antigen.
  • the composition confers an immune response against a fungal pathogen.
  • the composition confers an immune response against a viral pathogen.
  • the composition confers an immune response against a tumour antigen.
  • one of the at least two populations of T cells is enriched for CD137 expressing cells.
  • methods for producing a composition of the present disclosure are also provided.
  • the present disclosure provides a method for producing the composition described herein, comprising:
  • step b) repeating step a) for each population of cells in the composition
  • the isolated populations comprise activated T cells.
  • the activated T cells are isolated using an activation marker.
  • the activation marker is CD137.
  • the activation marker is CD154.
  • the population of isolated cells comprising T cells is enriched for CD 137 expressing cells.
  • each isolated CD 137 enriched T cell population comprises about 10-40% CD4+ cells, or about 10-20%, or about CD4+ cells, or about 20-30% CD4+ cells, or about 3-40% CD4+ cells, or about 50-100% CD4+ cells, or about 60- 100% CD4+ cells, or about 60-90% CD4+ cells.
  • each isolated CD137 enriched T cell population comprises about 60-70% CD4+ cells, such as about 60% CD4+ cells, or about 65% CD4+ cells, or about 70% CD4+ cells.
  • each isolated CD 137 enriched T cell population comprises about 70-80%) CD4+ cells, such as about 75% CD4+ cells, or about 80% CD4+ cells.
  • each isolated CD 137 enriched T cell population comprises about 80-90%o CD4+ cells, such as about 85% CD4+ cells, or about 90% CD4+ cells.
  • each isolated CD 137 enriched T cell population comprises about 90-100% CD4+ cells, such as about 95% CD4+ cells, or about 100% CD4+ cells.
  • each isolated CD 137 enriched T cell population comprises about 10-40% CD8+ cells, or about 10-20%, or about CD8+ cells, or about 20-30% CD8+ cells, or about 3-40% CD8+ cells, or about 50-100% CD8+ cells, or about 60- 100% CD8+ cells, or about 60-90% CD8+ cells.
  • each isolated CD137 enriched T cell population comprises about 60-70% CD8+ cells, such as about 60% CD8+ cells, or about 65% CD8+ cells, or about 70% CD8+ cells.
  • each isolated CD137 enriched T cell population comprises about 70-80%) CD8+ cells, such as about 75% CD8+ cells, or about 80% CD8+ cells.
  • each isolated CD 137 enriched T cell population comprises about 80-90% CD8+ cells, such as about 85% CD8+ cells, or about 90% CD8+ cells.
  • each isolated CD137 enriched T cell population comprises about 90-100% CD8+ cells, such as about 95% CD8+ cells, or about 100% CD8+ cells.
  • the method comprises expanding the isolated populations comprising T cells in vitro.
  • the isolated T cell populations are enriched for CD 137 expressing cells prior to in vitro expansion.
  • the isolated populations comprising T cells are expanded in vitro prior to combining in a defined ratio or defined number.
  • the method comprises expanding the population of cells comprising T cells in vitro prior to combining, for example, using one or more cytokines.
  • the method comprises contacting a population of cells comprising T cells with a fungal antigen, a viral antigen or a tumour antigen. In one example, the method comprises contacting each population of cells comprising T cells with a fungal antigen, a viral antigen or a tumour antigen. For example, the method of contacting is repeated for each population of T cells in the composition. In one example, the method comprises contacting the population(s) of cells comprising T cells with the fungal antigen, viral antigen or tumour antigen at least twice.
  • the method comprises isolating the populations comprising T cells following contacting with a fungal antigen, a viral antigen or a tumour antigen.
  • Methods of isolating populations of T cells will be apparent to the skilled person, or are described herein.
  • the method comprises combining the populations (i.e., at least two populations) of T cells in a defined number or a defined ratio to produce the composition of the present disclosure.
  • the population(s) comprising T cells are populations of peripheral blood mononuclear cells or G-CSF mobilized hematopoietic stem cells or bone marrow cells.
  • the method of the present disclosure comprises:
  • the method of the present disclosure comprises:
  • the method of the present disclosure comprises:
  • the method additionally comprises formulating the population(s) of T cells into a pharmaceutically acceptable carrier.
  • the present disclosure comprises a composition produced by the methods described herein.
  • the present disclosure provides a method of treating a subject in need thereof, the method comprising administering the composition of the present disclosure or the population of cells comprising T cells produced by the method of the present disclosure.
  • the method comprises administering an effective amount of the composition of the present disclosure or the population of cells comprising T cells produced by the method of the present disclosure, such as a therapeutically effective amount of the composition of the present disclosure or the population of cells comprising T cells produced by the method of the present disclosure.
  • the subject is undergoing or is about to commence or has completed chemotherapy and/or hematopoietic stem cell transplantation and/or immunoablation therapy and/or solid organ transplantation and/or has an inherited familial or congenital immunodeficiency syndrome and/or has an acquired immunodeficiency syndrome and/or is receiving or has received immunosuppressive therapy for an immune mediated disease.
  • the subject has completed chemotherapy, e.g., a subject who has received chemotherapy for a blood cancer, such as lymphoma or leukemia.
  • a blood cancer such as lymphoma or leukemia.
  • the subject has completed hematopoietic stem cell transplantation.
  • the subject is receiving immunosuppressive therapy following solid organ transplantation.
  • the subject is about to receive or has received a solid organ transplantation, e.g., transplantation of kidney, liver, pancreas, pancreatic islets, heart, lung, small bowel or other solid organ.
  • a solid organ transplantation e.g., transplantation of kidney, liver, pancreas, pancreatic islets, heart, lung, small bowel or other solid organ.
  • the subject is receiving or has received immunosuppressive therapy, antibody treatment or soluble receptor treatment or another immunomodulating treatment for an immune mediated disease, e.g., inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, hepatitis, glomerulonephritis and kidney failure, cancer, lymphoma, leukemia, myelodysplasia, myeloma.
  • an immune mediated disease e.g., inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, hepatitis, glomerulonephritis and kidney failure
  • the subject is suffering from a disease or condition.
  • the subject suffers from an inherited familial or congenital immunodeficiency, e.g., severe combined immune deficiency, common variable immunodeficiency, alymphocytosis, Wiskott Aldrich syndrome, ataxia telangiectasia, di George syndrome, leucocyte adhesion defects, immunoglobulin deficiency.
  • an inherited familial or congenital immunodeficiency e.g., severe combined immune deficiency, common variable immunodeficiency, alymphocytosis, Wiskott Aldrich syndrome, ataxia telangiectasia, di George syndrome, leucocyte adhesion defects, immunoglobulin deficiency.
  • the subject has acquired immunodeficiency through infection with the human immunodeficiency virus or another pathogenic organism that has led to incompetence of the immune system or is suffering from chronic relapsing fungal infections, e.g., chronic or relapsing oral or vaginal fungal infections such as candidiasis, chronic or relapsing fungal skin infection, chronic or relapsing fungal nail infections, chronic or relapsing fungal bronchial infections, chronic or relapsing fungal sinus infections, chronic or relapsing fungal myocardial infections, chronic or relapsing fungal cerebral infections, chronic or relapsing fungal bone infections, chronic or relapsing fungal liver infections, chronic or relapsing fungal kidney or bladder infection.
  • chronic relapsing fungal infections e.g., chronic or relapsing oral or vaginal fungal infections such as candidiasis, chronic or relapsing fungal skin infection, chronic or relapsing
  • the subject suffers from a cancer, such as a blood or bone marrow cancer
  • a cancer such as a blood or bone marrow cancer
  • the cancer includes multiple myeloma, leukemia, lymphoma, neuroblastoma, Ewing sarcoma, myelodysplastic syndromes and gliomas.
  • the disease or condition is a non-malignant condition, for example thalassemia, aplastic anemia, fanconi anemia and immune deficiency syndromes.
  • the condition or disease is associated with infection or graft-versus host disease.
  • the method comprises obtaining the cells from a subject and administering the composition to the same subject.
  • the T cells are autologous to the subject.
  • the method comprises obtaining cells from a fully HLA matched donor (i.e., a donor who has the same HLA antigens as the subject) and administering the composition to the subject.
  • a fully HLA matched donor i.e., a donor who has the same HLA antigens as the subject
  • the donor is a stem cell donor.
  • the cells are allogeneic, e.g., HLA-matched allogeneic cells.
  • the method comprises obtaining cells from a partially HLA matched donor (i.e., a donor who has one or more HLA antigens the same as the subject) and administering the composition to the subject.
  • the donor is a stem cell donor.
  • the cells are allogeneic, e.g., partially HLA-matched allogeneic cells.
  • the HLA antigen is HLA-A, HLA-B, HLA-C, HLA-DQ or HLA-DR.
  • the HLA antigen is HLA-DR.
  • the method comprises matching at least one HLA antigen in the cells to at least one HLA antigen in the subject and administering the composition to the subject.
  • the matching HLA antigen is the antigen presenting an infection or tumour antigen to the T cells in the subject.
  • the HLA antigen is the same for each population of cells in the composition.
  • the HLA antigen is different for different infection and tumor specificities.
  • the HLA antigen in each population of T cells in the final composition matches different HLA antigens in the subject depending on the infection and tumor specificity.
  • the shared HLA antigen is an HLA-DR antigen, or an HLA-A antigen, or an HLA-B antigen, or an HLA-C antigen, or an HLA-DP antigen, or an HLA-DQ antigen, or combinations thereof.
  • the T cells are non-autologous or allogeneic to the subject.
  • the term "obtaining cells" from a donor encompasses obtaining the cells from a bank of donor cells.
  • the cells are obtained from a cryopreserved bank of donor cells.
  • HLA-matched allogeneic cells or partially HLA-matched allogeneic cells are obtained from the bank and administered to the subject.
  • the banked cells are selected for a stem cell transplant recipient with an invasive fungal infection.
  • banked cells are obtained from a third party donor, stimulated with one or more fungal extracts, CD 137 selected and culture expanded with one or more cytokines.
  • at least one HLA-allele in the banked cells is matched to a HLA-DR allele in the stem cell transplant recipient (i.e., subject) to which the composition is administered.
  • the method comprises isolating T cells from multiple donors (e.g., healthy donors), identifying at least one HLA antigen in the T cells, producing a composition according to the present disclosure and banking the composition for future use.
  • the HLA antigen in the T cells matches the antigen presenting the infection or tumour antigen to the T cells in the subject.
  • the method further comprises matching at least one HLA antigen in the banked composition with at least one HLA antigen (i.e., the HLA antigen presenting an infection or tumor antigen) in the subject and administering the composition to the subject.
  • the HLA antigen is an HLA-DR antigen, or an HLA-A antigen, or an HLA-B antigen, or an HLA-C antigen, or an HLA-DP antigen, or an HLA-DQ antigen.
  • the present disclosure provides a bank comprising a plurality of compositions of the present disclosure.
  • the HLA is an HLA-DR antigen, or an HLA-A antigen, or an HLA-B antigen, or an HLA- C antigen, or a HLA-DP antigen, or an HLA-DQ antigen.
  • the present disclosure provides a method of treating a subject in need thereof, the method comprising determining an HLA antigen in the subject, matching the HLA antigen to an HLA antigen in T cells in a composition in the bank of the present disclosure and administering to the subject a composition comprising T cells having the same HLA antigen as that in the subject.
  • the HLA antigen of the T cell in the composition is the same as the HLA antigen presenting an infection or tumour antigen in the subject.
  • the HLA antigen of the T cell in the composition is the same as the HLA antigen presenting an infection or tumour antigen in the subject.
  • the HLA antigen of the T cell in the composition is the same as the HLA antigen present an infection or tumour antigen in the subject but is a different allelic subtype of the HLA antigen.
  • Figure 1 is a graphical characterisation of individual fungal T cell cultures.
  • Percentage of responding CD4+ cells from culture stimulated with a combination of A terreus and C krusei and H. rechallenged with A terreus, C h'usei or all fungal lysates and I. as for H. but rechallenged with all individual lysates and all lysates combined (showing percentage of cells producing TNFa, INFy and/or IL-17). Bars indicate means ⁇ SEM (n 3-6).
  • Figure 2 is a graphical characterisation of multipathogen T cell cultures initiated with simultaneous stimulation of cultures with antigens from CMV, EBV, Adenovirus and A fumigatus lysate.
  • C Proportion of IFNy producing CD4 + or CD8 + T cells specific for CMV, AdV or A. fumigatus.
  • Figure 3 is a graphical characterisation of individual viral and fungal T cell cultures.
  • A Expansion of T cell cultures stimulated with individual fungal or viral antigens followed by CD 137 selection and expansion. Data expressed as mean cell number ⁇ SEM.
  • Figure 4 is a graphical characterisation of WT1 T cell cultures
  • B WT1 specificity as measured by CD107 expression after restimulation of cultures generated in A and restimulated with WT1 peptide mixture. Data expressed as mean ⁇ SEM; p ⁇ 0.05).
  • C Proportion of IFN- ⁇ CD8 T cells specific for WT1.
  • Figure 5 is a graphical characterisation of T cell products generated from single T cell cultures pooled in defined ratios compared to cultures initiated with simultaneous stimulation with antigens from multiple pathogens. Percentages show responding cells from cultures following rechallenge with individual antigens.
  • Figure 6 is a graphical characterisation of multipathogen T cell cultures initiated by simultaneous stimulation with antigens from A. terreus and C. krusei, followed by CD137 selection and in vitro expansion.
  • C Exhaustion markers expression profile (PD-1, Tim-3, CTLA-4 and Lag-3).
  • T central memory CD45RA " 62L +
  • T terminal effector CD45RA + 62L “
  • T Naive CD45RA + 62L +
  • T effector memory CD45RA " 62L “
  • Figure 7 shows the percentage of different CD4 T helper subtypes present in the panfungal T cell product; T h l (CCR4 " CCR6 " CCR10 " CXCR3 + ), T h 2 (CCR4 + CCR6 “ CXCR3 “ ), T h 9 (CCR4 " CCR6 + ), T h 17 (CCR4 + CCR6 + CCR10 XCR3 " ), T h 22 (CCR4 + CCR6 + CCR10 + ), T h l/T h 17 (CCR4 " CCR6 + CXCR3 + ). Results are means of 3 experiments ( ⁇ s.e.m) and shown as percent of CD4 T cells.
  • FIG 8 shows results from an immunoblot analysis of cytokine in supernatants of fungus- specific T cells cultured for 24 h with non-pulsed DCs (control) or fungus- pulsed DCs (activated). Data (means ⁇ s.e.m of 2 experiments) are presented as relative density measurement (RDM) in activated fungus-specific T cells relative to control.
  • RDM relative density measurement
  • Figure 9 shows the cytokine expression profile of panfungal T cells cultured for 5 h with non-pulsed DCs or fungus-pulsed DCs. Results are means of 3 experiments ( ⁇ s.e.m) and shown as percentage of CD4 + T cells.
  • Figure 10 is a t-distribution stochastic neighbourhood embedding (t-SNE) plot showing the mapping of panfungal T cells cultured for 5 h with non-pulsed DCs or fungus-pulsed DCs. Responding fungus-specific T cells correspond to the cell cluster showing higher GM-CSF, IFN- ⁇ and TNF-a expression, indicative of a majority cells with similar phenotype.
  • t-SNE stochastic neighbourhood embedding
  • Figure 11 shows the percent of hyphal damage (assessed by XTT colorimetric assay) induced by T cells, white blood cells (WBC) and combination of both after 2 h incubation with germinated conidia from A. fumigatus. Results are means of 4 experiments ( ⁇ s.e.m) and shown as percent of hyphal damage relative to non-treated hyphae (*p ⁇ 0.05 and **p ⁇ 0.001 treatment versus none; Student's t-test).
  • Figure 12 is a graphical characterisation of multipathogen T cell cultures initiated by simultaneous stimulation with antigens from A. terreus and C. krusei, followed by CD 137 selection and in vitro expansion.
  • A. Inhibition of CD4+ production of TNF-a following incubation of dendritic cells pulsed with fungal lysate and fungus specific T cells in the presence of specific HLA DRBl, DP and DQ antibodies showing that the production of TNF-a is mediated principally via HLA DRBl .
  • CD4+ cells producing TNF-a following presentation of fungal antigen by DCs that are autologous to the fungus specific T cells, partially matched at HLA DRBl (ie, one of two HLA DRBl antigens fully allelic matched), partially matched with allele subtype mismatched (one of two HLA DRBl antigen matched but allelic mismatched).
  • C. - G Percentage of CD4+ panfungal T cells generated using starting populations of DRBl 01 :01, 04:01, 1 1 :01, 13:01 or 15:01 able to recognize fungal antigens presented through other allelic subtypes of each of those DRBl antigens.
  • Panfungal T cells generated using HLA DRBl 01 :01 T cells respond to fungal antigens presented on DCs expressing HLA DRBl 01 :02.
  • Panfungal T cells generated using HLA DRBl 04:01 T cells respond to fungal antigens presented on DCs expressing HLA DRBl 04:03, HLA DRBl 04:04 and HLA DRBl 04:05.
  • Panfungal T cells generated using HLA DRBl 11 :01 T cells respond to fungal antigens presented on DCs expressing HLA DRBl 11 :04.
  • F. Panfungal T cells generated using HLA DRBl 13:01 T cells respond to fungal antigens presented on DCs expressing HLA DRBl 13:02.
  • Panfungal T cells generated using HLA DRBl 15:01 T cells respond to fungal antigens presented on DCs expressing HLA DRBl 15:02. Results are means of 3 to 6 experiments ( ⁇ s.e.m) and shown as percent of CD4 T cells.
  • FIG. 13 shows that HLA DRBl homozygous B cell lymphoblastoid cell lines
  • B-LCL present fungal antigen to CD4+ fungus T cells and can be used for screening of HLA element of restriction.
  • TNF-a production by HLA DRBl heterozygous fungal T cells originally expanded with A, terreus and C. krusei
  • HLA DRBl homozygous B cell lymphoblastoid cell lines B-LCL
  • Results are means of 5 experiments ( ⁇ s.e.m) and shown as percent of CD4+ T cells.
  • Figure 14 shows the proportion of the top TCR- ⁇ sequences expressed in reactive A.
  • Figure 15 is a graphical characterisation of peripheral blood mononuclear cells from a healthy male control aged 47 years, and from a male patient aged 51 years with disseminated Scedosporium boydii and treated with a composition comprising T cells.
  • A. Plots show live cells gated and analysed as CD3 v CD20.
  • B. Plots show gated CD4+ cells as CD45RO v CD366 (Tim3) (upper row) and gated CD8+ cells as CD45RO v CD366 (Tim3) (lower row).
  • C. Plots show total live cells gated and analysed as CD3 v GM-CSF (upper row), CD3 v IL10 (middle row) and CD3 v IL4 (bottom row).
  • D. Plots show live cells sequentially gated as CD3-CD20-HLA DR+CD56- and analysed as CD14 v CD16.
  • T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity and, to a lesser degree the adaptive immune response. Generally, T cells are distinguished from other lymphocytes (e.g., B cells and natural killer cells) by the presence of T cell receptors (TCRs). T cells have diverse roles, which are accomplished by differentiation of distinct populations of T cells, recognizable by discrete gene expression profiles.
  • the term "reactive" shall be taken to mean a T cell that is responsive to an antigen stimulus.
  • it refers to a T cell having antigenic reactivity against fungi, virus or tumour.
  • fungi refers to a member of a large group of eukaryotic organisms that may include microorganisms, e.g., yeasts and moulds. These organisms may be classified as a kingdom of fungi, which is separate from plants, animals, and bacteria. One major difference between fungi and the others is that fungal cells have cell walls that contain chitin, unlike the cell walls of plants, which contain cellulose.
  • fungi may refer to fungi which may cause infection in humans and animals. Such fungi may also be referred to as “pathogenic fungi”.
  • extract shall be taken to refer to a fungal extract obtained by any extraction method, whether or not the crude extract has been fractionated or purified.
  • extract and “lysate” in relation to fungal extracts are used interchangeably herein.
  • water soluble lysate shall be taken to mean a fungal lysate that substantially dissolved in water under the conditions of temperature and concentration at which the lysate is to be used.
  • germinated spores as used herein shall be taken to mean a fungal spore that has been subjected to germination conditions.
  • the term "obtaining the lysate” shall be taken to include the process of homogenization of the germinated fungal spores and purification of the lysate.
  • the term "virus” or “viral” as used herein, refers to a small infectious agent that replicates only inside the living cells of other organisms.
  • viruses include cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (AdV), varicella zoster virus (VZV), influenza and BK virus (B V), John Cunningham (JC) virus, respiratory syncytial virus (RSV), parainfluenzae, rhinovirus, human metapneumovirus, herpes simplex virus (HSV) 1, HSV II, human herpes virus (HHV) 6, HHV 8, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, rotavirus, papillomavirus and parvovirus.
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • AdV adenovirus
  • VZV varicella zoster virus
  • influenza and BK virus B V
  • JC John Cunningham
  • RSV respiratory syncytial virus
  • RSV respiratory syncytial virus
  • viral antigen refers to a protein encoded by the viral genome or a peptide derived therefrom.
  • the viral antigen may be in the form of overlapping peptides from viral proteins, a lysate of virally infected cells.
  • the viral antigen may also be presented by or expressed by recombinant cells (e.g., cells genetically engineered with retrovirus, lentivirus or other vectors).
  • tumour antigen refers to a protein encoded by a tumour genome or a peptide derived therefrom.
  • the tumour antigen may be in the form of overlapping peptides from tumour proteins (e.g., a peptide pool).
  • tumour antigens include wilms tumour antigen (WT1), beta chain of human chorionic gonadotropin (hCG beta) antigen, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), CD19, CD20, CA-125, epithelial tumor antigen (ETA), abnormal products of ras, p53, glycosphingolipid GD2, prostatic acid phosphatase (PAP), preferentially expressed antigen in melanoma (PRAME), B melanoma antigen (BAGE), cancer-testis antigen (NY ESO-1), sarcoma antigen 1 (SAGE), helicase antigen (HAGE), cancer- germline antigen (GAGE), prostein (P501S), six-transmembrane epithelial antigen of the prostate (STEAP), Plu-1, human achaete-scute homolog-1 (hASHl), Cripto, Criptin, EGFRvIII antigen, Globo H antigen
  • the term "defined number” refers to a set number or amount of cells or population of cells in the composition. As the skilled person will be aware, when dealing with a cell based composition, the definition a specific number does not mean that exact number of cells is to be included in a composition. For example, a variation of up to about 10% is encompassed by this term.
  • the term "defined ratio” refers to the quantitative relationship between two or more amounts showing the number of times one value contains or is contained within the other.
  • the compositions described herein may comprise a defined ratio of two or more populations of T cells, each population being reactive to a particular antigen.
  • a specific ratio does not mean that exact ratio of cells is to be included in a composition. For example, a variation of up to about 10% is encompassed by this term.
  • enriched or “enrich” in the context of a cell population shall be taken to encompass a population of CD 137 T cells, including a population in which the number or percentage of CD 137 T cells is greater than the number or percentage in a naturally occurring cell population.
  • a CD 137 enriched population is made up of at least about 0.02% of said cells, or at least about 0.05% of said cells or at least about 0.1% of said cells or at least about 0.2% of said cells or at least about 0.5% of said cells or at least about 0.5% of said cells or at least about 0.8% of said cells or at least about 1% of said cells or at least about 2% of said cells or at least about 3% of said cells or at least about 4% of said cells or at least about 5% of said cells or at least about 10% of said cells or at least about 15% of said cells or at least about 20% of said cells or at least about 25% of said cells or at least about 30% of said cells or at least about 40% of said cells or at least about 50% of said cells or at least about 60% of said cells or at least about 70% of said cells or at least about 80%o of said cells or at least about 85% of said cells or at least about 90% of said cells or at least about 95% of said cells or at least about 97% of said cells or at least about 98% of said cells or at least about
  • mycoplasma refers to a genus of bacteria that lack a cell wall around their cell membrane. Mycoplasma can be parasitic or saprotrophic.
  • the term "effective amount” shall be taken to mean a sufficient quantity of the composition to confer a therapeutic or protective immune response against fungi in a subject.
  • the skilled artisan will be aware that such an amount will vary depending on, for example, the fungi and/or the particular subject and/or the type or severity of a condition being treated. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, for example, weight or number of population of cells of the composition of the present disclosure.
  • the term "confers" shall be taken to mean that administration of the composition of the present disclosure initiates an immune response in a subject.
  • therapeutic immune response shall be taken to mean that administration of the composition is sufficient to induce an immune response that results in the reduction or inhibition of one or more symptoms of the infection.
  • protective immune response shall be taken to mean that administration of the composition is sufficient to induce an immune response that is capable of reducing or inhibiting, via IgG antibody production or T cell activation or enhancement of the actions of innate immune effectors such as monocytes, macrophages and/or neutrophils, infection.
  • condition refers to a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.
  • treating include administering a cell or cells described herein to thereby reduce or eliminate at least one symptom of a specified condition or disease.
  • the term "subject" shall be taken to mean any animal, such as, a mammal.
  • the mammal is a human or non-human primate.
  • the mammal is a human.
  • the term "donor" refers to a subject from whom cells are collected, said cells being intended for the preparation of a bank according to the present disclosure or for future preparation of a composition according to the present disclosure.
  • the donor may be a stem cell donor from whom cells are collected as part of stem cell transplantation.
  • the donor may be a solid organ donor from whom cells are collected as part of an organ transplant.
  • autologous shall be taken to mean the cells are obtained from the subject that is undergoing treatment with the cells of the present disclosure.
  • non-autologous shall be taken to mean the cells are from a donor.
  • the cells are obtained from a subject that is different from the subject receiving the treatment with the cells of the present disclosure.
  • MHC major histocompatibility complex
  • WBCs white blood cells
  • HLA human leukocyte antigen
  • Exemplary fungi of the present disclosure include filamentous fungi, e.g., of the genera Aspergillus, Fusarium, mucor/zygomycetes (Rhizopus) and Scedosporium/Lomentospora species, and the yeast Candida.
  • the fungus is of the Aspergillus genera.
  • the fungus is yeast of the C. krusei genera.
  • the filamentous fungi include A. fumigatus, A. flavus, A. terreus, F. oxysporum, F. solani, R. oryzae and L. proliflcans.
  • the fungus is A. terreus.
  • Exemplary yeast of the present disclosure includes C. albicans and C. krusei. In one example, the yeast is C. krusei.
  • the fungus is isolated from the environment.
  • A. fumigatus, A. terreus and F. oxysporum are isolated from the environment.
  • the fungus is isolated from a clinical specimen.
  • A. fumigatus, A. terreus, F. oxysporum, F. solani, R. oryzae, L. proliflcans, C. albicans, C. krusei, G. glabrata are obtained from clinical specimens.
  • Methods for isolating fungal lysates from clinical specimens are known in the art and described, for example, in Braedel et al. British Journal of Haemotology, 125: 392-399, 2004 or Gaundar et al. Cytotherapy, 14: 1119-1130, 2012.
  • the fungus is obtained from a repository, such as the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • A. flavus is obtained from ATCC.
  • the A. flavus is strain ATCC-204304.
  • a T cell of the disclosure is reactive with a fungal or yeast spore, e.g., a cultured and/or germinated spore.
  • a fungal or yeast spore e.g., a cultured and/or germinated spore.
  • Methods for culturing and germinating fungal spores will be apparent to the skilled person.
  • fungi isolated from the environment, clinical specimens or the ATCC are sub-cultured on potato dextrose agar plates for 3-7 days. Spores are removed by washing and separated from hyphal fragments by passing through 40 ⁇ or 60 ⁇ pore filters. Spores are germinated in potato dextrose medium for 16-72 hours at 25-37°C with agitation at 200rpm.
  • extracts or lysates of fungi or spores are produced. Methods for purification of fungal lysates or extracts will be apparent to the skilled person.
  • the germinated spores of the fungi are lysed.
  • the lysates are obtained by homogenizing germinated spores of fungi. As exemplified herein, following germination of the spores, the mycelial mat is washed with sterile water and then homogenized using 0.5mm zirconia-silica beads in a mini-beadbeater-8 cell homogenizer. Fungal lysates are clarified by centrifugation and passed through 0.22 ⁇ sterile filters.
  • BCA bicinchoninic acid
  • the lysate is not contaminated with bacteria or fungi.
  • the sterility of lysates is confirmed by the absence of bacterial or fungal growth after incubation for two weeks at 30°C in liquid culture medium.
  • viruses of the present disclosure include cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (AdV), varicella zoster virus (VZV), influenza and BK virus (B V), John Cunningham (JC) virus, respiratory syncytial virus (RSV), parainfluenzae, rhinovirus, human metapneumo virus, herpes simplex virus (HSV) 1, HSV II, human herpes virus (HHV) 6, HHV 8, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, rotavirus, papillomavirus and parvovirus.
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • AdV adenovirus
  • VZV varicella zoster virus
  • influenza and BK virus B V
  • JC John Cunningham
  • RSV respiratory syncytial virus
  • RSV respiratory syncytial
  • the viral antigen is in the form of overlapping peptides from viral proteins or a lysate of virally infected cells.
  • the viral antigen is a peptide pool that consists mainly of 15-mer peptides with 11-amino acid overlap (e.g., a PepTivator®).
  • the viral antigen is presented by or expressed by recombinant cells (e.g., cells genetically engineered with retrovirus, lentivirus or other vectors).
  • tumour antigens of the present disclosure include wilms tumour antigen (WT1), beta chain of human chorionic gonadotropin (hCG beta) antigen, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), CD19, CD20, CA-125, epithelial tumor antigen (ETA), abnormal products of ras, p53, glycosphingolipid GD2, prostatic acid phosphatase (PAP), preferentially expressed antigen in melanoma (PRAME), B melanoma antigen (BAGE), cancer-testis antigen (NY ESO-1), sarcoma antigen 1 (SAGE), helicase antigen (HAGE), cancer-germline antigen (GAGE), prostein (P501S), six-transmembrane epithelial antigen of the prostate (STEAP), Plu-1, human achaete-scute homolog-1 (hASHl), Cripto, Criptin, EGFRvIII antigen,
  • the tumour antigen is in the form of overlapping peptides from tumour proteins.
  • the tumour antigen is a peptide pool that consists mainly of 15-mer peptides with 11-amino acid overlap (e.g., a PepTivator®).
  • T cell populations e.g., T cell populations
  • T cells are isolated from peripheral blood mononuclear cells (PBMCs), cord blood, G-CSF mobilized hematopoietic progenitor cells (HPCs), bone marrow, splenic tissue, thymic tissue and lymph nodes.
  • PBMCs peripheral blood mononuclear cells
  • HPCs G-CSF mobilized hematopoietic progenitor cells
  • the T cells are isolated from PBMCs.
  • T cells are isolated from G-CSF mobilised HPCs.
  • T cells are isolated from bone marrow.
  • PBMC are isolated from heparinised blood by gradient centrifugation over Ficoll-paque.
  • the donors of peripheral blood are immunologically normal undergoing therapeutic venesection.
  • 300- 500mL of peripheral blood is collected for isolation of PBMC.
  • the PBMC are cryopreserved in 10% dimethylsulfoxide (DMSO) solution.
  • DMSO dimethylsulfoxide
  • monocytes are isolated following mobilisation of stem cells by administration of granulocyte-colony stimulating factor (G-CSF).
  • G-CSF granulocyte-colony stimulating factor
  • donors of stem cells are healthy individuals donating for allogeneic hematopoietic stem cell transplantation.
  • the monocytes are freshly isolated.
  • the PMSC or monocytes are cryopreserved in 10% DMSO solution.
  • monocytes are isolated following harvest of bone marrow.
  • donors of bone marrow are healthy individuals donating for allogeneic hematopoietic stem cell transplantation.
  • the bone marrow mononuclear cells are freshly isolated.
  • the bone marrow mononuclear cells are cryopreserved in 10% DMSO solution.
  • HPC are isolated by washing in phosphate buffered saline containing 1% human albumin. In one example, the HPC are freshly isolated. In another example, the HPC are cryopreserved in 10% DMSO solution.
  • Exemplary T cell populations include naive T cells, T helper cells (3 ⁇ 4 cells), terminally differentiated effector T cells (T ef cells), effector memory T cells (T em cells), central memory T cells (T cm cells), cytotoxic T cells (CTLs) and regulatory T cells (T reg cells).
  • T cell populations are at least 98% T cells and less than 2% B cells and monocytes. In one example, the T cells express at least CD3 and/or CD4.
  • Exemplary T cells are reactive with a fungal antigen, a viral antigen or a tumour antigen.
  • the antigen-specific T cells are produced with G-CSF mobilised HSCs exposed to a fungal antigen, a viral antigen or a tumour antigen.
  • the T cells are produced with G-CSF mobilised HSCs exposed to a fungal extract of C. krusei or a fungal extract of A. terreus.
  • the T cells are produced with G-CSF mobilised HSCs exposed to a viral antigen from cytomegalovirus or Epstein-Barr virus or varicella zoster virus or adenovirus.
  • the T cells are produced with G-CSF mobilised HSCs exposed to a Wilms tumour 1 antigen
  • the T cells are stimulated by culturing with direct addition of the fungal antigen, viral antigen or tumour antigen.
  • the fungal extract is added at a concentration of l( ⁇ g/ml.
  • the viral antigen or tumour antigen is added in the form of a synthetic peptide pool.
  • the peptide pool is added at a concentration of 1 ⁇ g/ml.
  • the T cells are enriched for CD 137 expressing cells following antigen stimulation.
  • the T cells are selected for using an antibody against CD137.
  • the T cells are selected for using an immunomagnetic bead enrichment system (e.g., CliniMACS®).
  • the T cells are stimulated at least once.
  • the T cells are stimulated at least twice.
  • the T cells are stimulated twice.
  • Exemplary T cells are culture expanded following CD 137 enrichment.
  • the T cells are expanded in cell culture.
  • Cells of the disclosure can be maintained under standard cell culture conditions.
  • Cell cultures can be incubated at about 37°C in a humidified incubator.
  • Cell culture conditions can vary considerably for the cells of the present disclosure.
  • the cells are maintained in an environment suitable for cell growth, e.g., comprising 5% 0 2 , 10% C0 2 , 85% N 2 or comprising 10% C0 2 in air.
  • the T cells are co-cultured with CD137 negative cells.
  • the CD137 negative cells have been irradiated prior to co-culture.
  • the T cells are expanded with one or more cytokines.
  • the T cells are expanded with interleukin-2 (IL-2).
  • the T cells are expanded with interleukin-15 (IL-15).
  • the T cells are expanded with interleukin-7 (IL-7).
  • the cells are expanded with IL-2, IL-15 and IL-7.
  • the T cells are cultured in AIM-VTM medium or any other appropriate cell culture medium known in the art.
  • Other appropriate media include, Dulbecco's Minimal Essential Medium (DMEM), MCDB, Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM) and Roswell Park Memorial Institute (RPMI) 1640 medium.
  • DMEM Dulbecco's Minimal Essential Medium
  • MEM Minimal Essential Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • RPMI Roswell Park Memorial Institute
  • the AIM-VTM medium is supplemented with 20U/ml IL-2, lOng/ml IL-15 and lOng/ml IL-7.
  • the AIM-VTM medium is supplemented with 10% heat inactivated autologous plasma.
  • the cultures are replenished about 2, or about 3 times per week using fresh medium containing the appropriate concentration of cytokines.
  • Exemplary T cell cultures are maintained for a total of about 12-21 days.
  • cell numbers are determined by counting in a haemocytometer.
  • the T cells express Thl cytokines.
  • the T cells express a cytokine selected from the group consisting of TNFa, IFNy, IL-17, RANTES, IL-2, ⁇ - ⁇ , ⁇ - ⁇ , IL-5, IL13, GM-CSF, IL-8 and combinations thereof.
  • the T cells are negative for bacterial and fungal contamination.
  • the T cells are negative for bacterial and fungal contamination for a period of at least 5 days.
  • the T cells are negative for Mycoplasma contamination.
  • the T cells are non-reactive for infectious disease markers, such as, the presence of endotoxin, hepatitis viruses A, B or C, human immunodeficiency viruses that can be present in blood. Testing for Activity
  • the lymphoproliferative activity of the fungal lysate is assessed.
  • One method for assessing the activity of fungal lysates on lymphoid cells is using a 3 H-thymidine incorporation assay.
  • a 3 H-thymidine incorporation assay may involve the stimulation of PBMC (2xl0 5 ⁇ 1 ⁇ 8/200 ⁇ 1) with ⁇ 0 ⁇ iglm ⁇ of individual fungal lysate.
  • IL-2 25U/ml
  • lysate from K562 cells (10 ⁇ g/ml) may be used as an irrelevant control.
  • the lymphoproliferative activity may be assessed by addition of one ⁇ of thymidine followed by overnight incubation at 37°C, lysing of cells and harvesting of the cellular material onto filter mats.
  • 3 H-thymidine incorporation can be assessed on a MicrobetaTM2 plate reader.
  • the reagents for determining lymphoproliferative activity are commercially available from Perkin Elmer.
  • the T cells are phenotyped.
  • One method of phenotyping the T cells is staining the cells with fluorophore- conjugated antibodies to human CD3 (SK7), CD4 (RPA-T4), CD 8 (SKI), CD19 (SJ25C1), CD14 ( ⁇ 9), CD56 (NCAM16.2), V52TCR (B6), CD45RO (UCHL1), CD45RA (HI100), CD62L (Dreg56), CD25 (2A3), CD28 (L293), CD154 (TRAP1), CD161 (DX12) (all from BD; clones indicated in parentheses), Foxp3 (259D) (Biolegend) and CCR4 (205410) (R&D Systems).
  • the proliferative activity of the cultured T cells is assessed.
  • One method of assessing the proliferative activity of cultured T cells is to stain the cells with 1.5 ⁇ carboxyfluorescein succinimidyl ester (CFSE) for 10 min at 37°C then wash and culture the cells with individual antigen. The loss of CFSE fluorescence in cells after 7 days is then assessed by flow cytometry following co-staining with CD4 and CD8 antibodies.
  • CFSE carboxyfluorescein succinimidyl ester
  • the antifungal activity of the T cells is assessed.
  • One method of assessing the antifungal activity is using a calorimetric assay with 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tertrazolium-5-carboxyanilide sodium salt (XTT) and 2-methyl-l,4-napthoquinone (menadione).
  • XTT 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tertrazolium-5-carboxyanilide sodium salt
  • menadione 2-methyl-l,4-napthoquinone
  • PBMC white blood cells
  • WBC white blood cells
  • fungus-specific T cells can then be added to appropriate wells at an effector to target ratio of 5:1 (5xl0 4 of each cell type).
  • cells are lysed by washing twice in sterile water.
  • the hyphal masses are resuspended in 200 ⁇ 1 sterile water and then ⁇ of XTT- menadione reagent is added to each well. The plate is vortexed gently then incubated at 37°C for 2 hours.
  • % HD (1-X/C) x 100, where X is the absorbance of test well and C is the absorbance of the control wells with hyphae only.
  • the levels of cytokines in supernatants of antigen-stimulated T cells is assessed.
  • cytometric bead array One method of assessing the levels of cytokines in supernatants of antigen- stimulated T cells is using a cytometric bead array. Such a process may involve stimulating cultured T cells with one or more or all fungal lysates or viral or tumour antigens (or no stimulation control). The levels of TNF , IFNy, IL-2, regulated upon activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-l, macrophage inhibitory protein ( ⁇ )- ⁇ , IL-4, IL-8 and IL-17 in the cell-free supernatants can be measured after 5 hours of stimulation by cytometric bead array.
  • RANTES normal T cell expressed and secreted
  • MCP monocyte chemoattractant protein
  • ⁇ macrophage inhibitory protein
  • a kit for performing the cytometric bead array is commercially available from
  • the specificity of antigen-stimulated T cell cultures is assessed.
  • One method of assessing the specificity of antigen-stimulated T cell cultures is using flow cytometric analysis. Such a process may involve re-stimulating T cell cultures with antigen and incubating re-stimulated cells in the presence of anti- CD 107a/b antibodies (to measure cytotoxic activity) for 5 hours and the co-stimulatory molecules CD28 and CD49d. Following 1 hour, monensin and brefeldin is added to allow intracellular accumulation of cytokines. Cells are then stained with antibodies to CD3, CD4 and CD8. After fixation and permeabilization, intracellular cytokines are stained with antibodies to IFNy and TNFa. Cells are acquired and analysed on a fluorescence-activated cell sorting (FACS) machine (e.g., BD FACS Canto 2 using FACS diva software).
  • FACS fluorescence-activated cell sorting
  • T cell compositions The composition of the present disclosure is useful for parenteral administration for prophylactic or for therapeutic treatment of, e.g., superficial or invasive fungal disease, a viral infection, cancer, leukemia, lymphoma or other malignant disease.
  • the T cells are isolated following exposure to a fungal antigen or to a viral antigen or to a tumour antigen, and formulating the population(s) of cells into a pharmaceutically acceptable carrier.
  • the composition comprises one or more population(s) of T cells each population reactive with a fungal antigen.
  • the composition comprises one or more population(s) of T cells each population reactive with a viral antigen.
  • the viral antigen comprises overlapping peptides from viral proteins, a lysate of virally infected cells, cells genetically engineered with retrovirus, lentivirus or other vectors to express a viral protein.
  • the composition comprises one or more population(s) of T cells each population reactive with a tumour antigen.
  • the tumour antigen comprises overlapping peptides from tumour proteins, a lysate of tumour cells, cells genetically engineered to express a tumour protein.
  • the T cells are reactive with a fungal antigen from a fungal extract selected from the group consisting of Aspergillus terreus, Candida krusei, Rhizopus oryzae, A. fumigatus, A. flavus, A. terreus, C. albicans, C. krusei, F. oxysporum, F. solani and L. prolificans.
  • a fungal antigen from a fungal extract selected from the group consisting of Aspergillus terreus, Candida krusei, Rhizopus oryzae, A. fumigatus, A. flavus, A. terreus, C. albicans, C. krusei, F. oxysporum, F. solani and L. prolificans.
  • the T cells are reactive with a viral antigen from a virus selected from the group consisting of CMV, EBV, AdV, VZV, influenza, BK virus, JC virus, RSV, parainfluenzae, rhinovirus, human metapneumovirus, HSV 1, HSV II, HHV6, HHV8, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, rotavirus, papillomavirus and parvovirus.
  • a virus selected from the group consisting of CMV, EBV, AdV, VZV, influenza, BK virus, JC virus, RSV, parainfluenzae, rhinovirus, human metapneumovirus, HSV 1, HSV II, HHV6, HHV8, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, rotavirus, papillomavirus and parvovirus.
  • the T cells are reactive with a tumour antigen selected from the group consisting of Wilms tumour antigen (WT1), beta chain of human chorionic gonadotropin (hCG beta) antigen, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), CD19, CD20, CA-125, epithelial tumor antigen (ETA), abnormal products of ras, p53, glycosphingolipid GD2, prostatic acid phosphatase (PAP), preferentially expressed antigen in melanoma (PRAME), B melanoma antigen (BAGE), cancer-testis antigen (NY ESO-1), sarcoma antigen 1 (SAGE), helicase antigen (HAGE), cancer- germline antigen (GAGE), prostein (P501S), six-transmembrane epithelial antigen of the prostate (STEAP), Plu-1, human achaete-scute homolog-1 (hASHl), Cripto, Criptin,
  • the composition comprises at least two populations of T cells each population reactive with a fungal antigen, a viral antigen or a tumour antigen.
  • the composition comprises T cells reactive with fungal antigens only.
  • the composition comprises T cells reactive with viral antigens only.
  • the composition comprises T cells reactive with tumour antigens only.
  • the composition comprises one or more population(s) of T cells each population reactive with a fungal antigen and one or more population(s) of T cells each population reactive with a viral antigen.
  • the composition comprises one or more population(s) of T cells each population reactive with a fungal antigen and one or more population(s) of T cells each population reactive with a tumour antigen.
  • the composition comprises one or more population(s) of T cells each population reactive with a viral antigen and one or more population(s) of T cells each population reactive with a tumour antigen.
  • the composition comprises one or more population(s) of T cells each population reactive with a fungal antigen, one or more population(s) of T cells each population reactive with a viral antigen and one ore more population(s) of T cells each population reactive with a tumour antigen.
  • composition comprises T cell products that meet the following release criteria:
  • the cells are autologous to the subject receiving the treatment.
  • the cells are non-autologous to the subject.
  • Formulation of the composition to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion) selected.
  • An appropriate pharmaceutical composition comprising the composition of the present disclosure to be administered can be prepared in a physiologically acceptable carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • aqueous carriers include water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980).
  • the compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.
  • the optimum concentration of cell populations in the chosen medium can be determined empirically, according to procedures well known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.
  • the composition comprises a defined number or defined ratio of
  • the composition comprises an equal number of T cells in each population.
  • each population is combined in a ratio of between 1:1 and 1 :99.
  • each population is combined in a ratio of about 1:1 to about 1:3.
  • the defined number or defined ratio is based on a subject's clinical situation or clinical diagnosis.
  • the number or ratio of cells in a population is based on the perceived likelihood of severe infection with varying types of transplant.
  • a subject undergoing cord blood transplantation can receive a product with specificities for all available pathogens, whilst a subject undergoing a matched sibling transplant can receive only T cell populations targeting pathogens based on the reduced likelihood of opportunistic infection following transplant.
  • a subject with features of a specific infection that have or have not been confirmed by diagnostic investigations can receive a higher number or ratio of fungal specific T cells in the composition due to the likelihood of systemic fungal infection.
  • a subject with a history of a known infection prior to a planned or unplanned period of immunosuppression can receive a higher number or ratio of T cells specific for that infection as part of a composition.
  • the defined number or defined ratio is based on a subject's serology or infectious status.
  • a subject seropositive and/or viral nucleic acid test positive for a pathogen can receive a composition comprising T cells reactive with the specific pathogen.
  • the defined number or defined ratio is based on a subject's previous serology or infectious status.
  • a composition can be based on the detection of specific pathogens detected in a period prior to administration to target specific infections likely to become clinically apparent.
  • the defined number or defined ratio is based on a known profile for an infection. For example, if a pathogen is commonly associated with a disease or condition, T cells reactive with the pathogen can be included in the composition. In one example, the defined number or defined ratio is based on the age of patient undergoing allogeneic stem cell transplant. For example, a paediatric patient is more likely to develop systemic adenovirus infection and thus is more likely to receive a composition with a high number or ratio of adenovirus specific T cells.
  • the defined number or defined ratio is based on a known diagnosis of a tumor.
  • a patient with acute myeloid leukemia expressing the WT1 tumor antigen can receive a composition comprising an increased number or ratio of WT1 specific T cells.
  • a patient with acute lymphoblastic leukemia expressing CD 19 can receive a composition comprising an increased number or ratio of CD19 targeted T cells (e.g., CD19 chimeric antigen receptor bearing T cells).
  • compositions of the disclosure are those large enough to produce the desired effect.
  • composition comprises an amount sufficient to confer a therapeutic or protective immune response in the subject.
  • the dosage should not be so large as to cause adverse side effects, such as hyper viscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.
  • Dosage can vary from about 1 x 10 3 cells/kg to about 1 x 10 10 cells/kg.
  • the composition can comprise about 1 x 10 3 cell/kg to about 1 x 10 4 cells/kg, or about 1 x 10 4 cell/kg to about 1 x 10 s , or about 1 x 10 5 cell/kg to about 1 x 10 6 , or about 1 x 10 6 cell/kg to about 1 x 10 7 , or about 1 x 10 7 cell/kg to about 1 x 10 8 , or about 1 x 10 8 cell/kg to about 1 x 10 9 , or about 1 x 10 9 cell/kg to about 1 x 10 10 .
  • Dosage can vary from about 1 x 10 5 cells/m 2 to about 1 x 10 10 cells/m 2 .
  • about 1 x 10 5 cells/m to about 1 x 10 cells/m or about 1 x 10 cells/m to about 1 x 10 cells/m
  • about 1 x 10 cells/m to about 1 x 10 cells/m or about 1 x 10 cells/m to about 1 x 10 cells/m to about 1 x
  • 10 cells/m or about 1 x 10 cells/m to about 1 x 10 cells/m .
  • each population in the composition can vary from about 1 x 10 cells/kg to about 1 x 10 cells/kg.
  • each population in the composition can comprise about 1 x 10 3 cell/kg to about 1 x 10 4 cells/kg, or about 1 x 10 4 cell/kg to about 1 x 10 5 , or about 1 x 10 5 cell/kg to about 1 x 10 6 , or about 1 x 10 6 cell/kg to about 1 x 10 7 , or about 1 x 10 7 cell/kg to about 1 x 10 8 , or about 1 x 10 8 cell/kg to about 1 x 10 9 , or about 1 x 10 9 cell/kg to about 1 x 10 10 .
  • Dosage can vary from about 1 x 10 s cells/m 2 to about 1 x 10 10 cells/m 2 .
  • about 1 x 10 5 cells/m to about 1 x 10 cells/m or about 1 x 10 cells/m to about 1 x 10 cells/m , or
  • 10 cells/m or about 1 x 10 cells/m to about 1 x 10 cells/m .
  • the dosage may be administered in one or more dose administrations.
  • the dosage can be repeated at least once.
  • the dosage is repeated at intervals depending on the immune state of the subject and the response to the previous infusion.
  • the repeat dosage(s) need not be the same as previous dosage(s), e.g., it could be increased or decreased.
  • the composition is administered intravenously.
  • multiple doses may be administered.
  • increasing doses may be administered.
  • administration of the T cell population to a subject confers a therapeutic immune response against fungi or a virus or a tumour.
  • administration of the T cell population to a subject confers a protective immune response against fungi or a virus or a tumour.
  • the subject is suffering from a disease or condition.
  • the disease or condition is a cancer, such as a blood or bone marrow cancer, for example the cancer includes multiple myeloma, leukemia, lymphoma, neuroblastoma, Ewing sarcoma, myelodysplastic syndromes and gliomas.
  • the disease or condition is a non-malignant condition, for example thalassemia, aplastic anemia, fanconi anemia and immune deficiency syndromes.
  • the condition or disease is associated with infection or graft-versus host disease.
  • the subject is undergoing treatment for a disease or condition. In one example, the subject is about to commence or has completed treatment for a disease or condition. In one example, the treatment is chemotherapy, hematopoietic stem cell transplantation or immunoablation. For example, the subject is undergoing or about to commence or has completed chemotherapy and/or hematopoietic stem cell transplantation and/or immunoablation therapy.
  • the subject is about to receive or has received transplantation of a solid organ such as a kidney, liver, pancreas, pancreatic islets, heart, lungs, small bowel or other solid organ.
  • a solid organ such as a kidney, liver, pancreas, pancreatic islets, heart, lungs, small bowel or other solid organ.
  • the subject is receiving or has received immunosuppressive drug treatment or antibody treatment or soluble receptor treatment or another immunomodulating treatment for a disease such as, but not limited to, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, hepatitis, glomerulonephritis and kidney failure, cancer, lymphoma, leukemia, myelodysplasia, myeloma.
  • a disease such as, but not limited to, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, hepatitis, glomerulonephritis and kidney failure, cancer, lymphoma, leukemia, myelodysplasia, myeloma.
  • the subject has inherited or been born with a deficiency of the immune system such as, but not limited to, severe combined immune deficiency, common variable immunodeficiency, alymphocytosis, Wiskott Aldrich syndrome, ataxia telangiectasia, di George syndrome, leucocyte adhesion defects, immunoglobulin deficiency.
  • a deficiency of the immune system such as, but not limited to, severe combined immune deficiency, common variable immunodeficiency, alymphocytosis, Wiskott Aldrich syndrome, ataxia telangiectasia, di George syndrome, leucocyte adhesion defects, immunoglobulin deficiency.
  • the subject has an acquired immunodeficiency through infection with the human immunodeficiency virus or another pathogenic organism that has led to incompetence of the immune system.
  • the subject is suffering from chronic relapsing fungal infections such as, but not limited to, chronic or relapsing oral or vulvovaginal candidiasis, chronic or relapsing fungal skin infection, chronic or relapsing fungal nail infections, chronic or relapsing fungal bronchial infections, chronic or relapsing fungal sinus infections, chronic or relapsing fungal myocardial infections, chronic or relapsing fungal cerebral infections, chronic or relapsing fungal bone infections, chronic or relapsing fungal liver infections, chronic or relapsmg fungal kidney or bladder infection.
  • chronic relapsing fungal infections such as, but not limited to, chronic or relapsing oral or vulvovaginal candidiasis, chronic or relapsing fungal skin infection, chronic or relapsing fungal nail infections, chronic or relapsing fungal bronchial infections, chronic or relapsing fungal sinus infections, chronic or
  • the cells of the present disclosure are HLA-allele phenotyped.
  • the cells are partially HLA-allele phenotyped.
  • the cells have alleles selected from major HLA, such as any Class I, II or III HLA, minor HLA, and non-polymorphic alleles, such as any member of the CD1 family members.
  • Major HLA alleles may more specifically be selected from any class I HLA such as HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A11 , HLA-A28, HLA-A29, HLA-A32, HLA-B15, HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B14, HLA-B18, HLA-B35, HLA-B40, HLA-C group 1, HLA-C group 2 for example, any class II HLA- DPB9, HLA-DPB11, HLA-DPB35, HLA-DPB55, HLA-DPB56, HLA-DPB69 HLA- DPB84 HLA-DPB 87, HLA-DRB 1 , HLA-DQA1 , HLA-DQB1 , or any class III HLA.
  • the knowledge of a HLA phenotype can facilitate subsequent selection of cells for the preparation of the composition of the present disclosure.
  • At least one class I HLA is phenotyped.
  • at least one HLA- A, HLA-B or HLA-C is phenotyped.
  • At least one class II HLA is phenotyped.
  • at least one of HLA-DR, HLA-DP or HLA-DQ is phenotyped.
  • At least one HLA-allele in the cells of the present disclosure is matched to at least one HLA-allele in the subject to which the composition is administered.
  • the matched HLA antigen is the antigen presenting an infection or tumour antigen to the T cells in the subject.
  • at least one class I HLA is matched.
  • at least one of HLA- A, HLA-B and HLA-C is matched.
  • at least one class II HLA is matched.
  • at least one of HLA-DR, HLA-DP and HLA-DQ is matched.
  • the HLA antigen is the same for each population of cells in the composition.
  • the phenotype of the HLA antigen in the cells of the present disclosure is matched to a HLA antigen in the subject to which the composition is administered.
  • the method of treating a subject comprises determining an HLA antigen in the subject, matching the HLA antigen to an HLA antigen in T cells in a composition in the bank and administering to the subject a composition comprising T cells having the same HLA antigen as that in the subject.
  • the HLA is different for different infection and tumor specificities.
  • the HLA antigen in each population of T cells in the final composition matches different HLA antigen in the subject depending on the infection and tumor specificity.
  • the HLA antigen is an HLA-DR antigen, or an HLA-A antigen, or an HLA-B antigen, or an HLA-C antigen, or an HLA-DP antigen, or an HLA-DQ antigen, or a combinations thereof.
  • the method additionally comprises testing the composition for anti-fungal activity.
  • the method comprises testing the composition for activity against a fungus with which a subject is infected. Banking of T cells
  • a plurality of T cell compositions are in a bank.
  • the cells for use in the present disclosure may be "banked" for future use, at a cell bank or depository or storage facility, or any place where such as cells are kept cryopreserved, e.g., in liquid nitrogen, for safekeeping.
  • appropriate computer systems can be used for data processing, to maintain records relating to donor information and to ensure rapid and efficient retrieval of cells from the storage repositories.
  • each of the storage containers can be tagged with positive identification based on a distinctive property associated with the donor, lines or cell type, prior to storing in a bank according to the disclosure.
  • DNA genetic fingerprint and HLA typing may be used with secured identification mechanism such as acceptable methods using microchips, magnetic strip, and/or bar code labels. This identification step may be included in the banking process.
  • the HLA is a HLA-DR antigen or an HLA- A antigen, or an HLA-B antigen, or an HLA-C antigen, or an HLA- DP antigen, or an HLA-DQ antigen.
  • the specificity of the T cells i.e., the target antigen
  • the target antigen is a viral antigen or a fungal antigen or a tumor antigen.
  • the HLA antigen presenting the target antigen to the T cell has been identified.
  • the HLA antigen is HLA-0201 presenting a CMV peptide NLVPMVATV.
  • the HLA antigen is HLA-DR04 presenting a fungal antigen.
  • the HLA antigen is HLA-B7 presenting an EBV antigen.
  • Certain diseases may require cell therapy that includes a series of repeated treatments.
  • the population of cells may be extracted from the bank and increased by cellular expansion before preparation of the pharmaceutical composition and administration to the subject.
  • Suitable cells for use in the preparation of the composition of the present disclosure may be obtained from existing cell banks, or may be directly collected from one or more donor subjects and later banked. In one example, cells are collected from healthy subjects. For example, cells from tissues that are non-essential to the subject may also be appropriate as they reduce the risk of induction of autoimmune disease.
  • Standards for donor selection may include one or more of the following considerations prior to collection, such as (a) absence of specific disease; (b) specific or general diseases; (c) parameters of the donor relating to certain diseases, for example a certain age, certain physical conditions and/or symptoms, with respect to certain specific diseases, with respect to certain prior treatment history and/or preventive treatment, etc.; (d) whether the donor fits into one or more established statistical and/or demographic models or profiles (e.g., statistically unlikely to acquire certain diseases); and (e) whether the donor is in a certain acceptable health condition as perceived based on prevailing medical practices, etc.
  • the cells are collected by apheresis from donor's peripheral blood, processed (to optimise the quantity and quality of the collected cells) and, optionally cryogenically preserved or maintained in culture under suitable conditions.
  • the donor is a stem cell donor.
  • the cells are collected by apheresis as part of the stem cell donation.
  • the cells are collected after administration of G-CSF to the donor alone or in combination with chemotherapy or a stem cell mobilising agent.
  • the cells are collected by bone marrow harvest.
  • the cells are collected by apheresis from the donor's peripheral blood or from the bone marrow by marrow harvest and are used for the preparation of the composition if the number of cells collected exceeds the number required for the purposes of stem cell transplantation.
  • the cells collected for the preparation of the composition are in excess of the cells required for stem cell transplantation.
  • the collected cells can be aliquoted into defined dosage fractions.
  • the cells may be stored under any appropriate conditions, such as in culture or in a cryopreserved state. Methods of cell storage will be apparent to the skilled person. For example, cryopreservation of cells can be achieved using a variety of cryoprotecting agents, such as DMSO.
  • the cells may be cryopreserved at different stages.
  • the cells may be cryopreserved as PBMC, isolated monocytes, isolated CD137 selected cells, and CD137 selected cells after about 1 to 2 weeks culture.
  • T cells are cryopreserved for adoptive T cell transfer.
  • a freezing mix containing 40% saline, 40% Albumex20 and 20% DMSO is prepared.
  • the saline is added to the DMSO and chilled before adding the Albumex20.
  • the freezing mix is kept chilled until required.
  • the cells for cryopreservation are resuspended, pooled and mixed thoroughly.
  • the cells are counted using a haemocytometer and the cell concentration and total cell viability is determined.
  • the cells are spun at 1400rpm for 5 mins and lOmls of the supernatant is removed for sterility and mycoplasma testing. The remaining supernatant is discarded.
  • the cells are washed with up to 200ml of 0.9% saline supplemented with Albumex20 and spun at 1400rpm for 5 mins.
  • the cells are resuspended in 0.9% saline at a concentration of 2xl0 7 cells/ml.
  • the number of bags and quality assurance samples to be cryopreserved is determined. An equal volume of freezing mix is added to the T lymphocyte suspension and mixed. The required volume of cells is transferred into cryopreservation bags and/or vials. The bags and vials are immediately placed into pre-cooled rate controlled freezers to begin cryopreservation.
  • Fungi were sub-cultured on potato dextrose agar plates for 3-7 days. Spores were removed by washing and separated from hyphal fragments by filtration (40 ⁇ or 60 ⁇ pore filters). Spores were germinated in potato dextrose medium for 16-72 hours at 25-37°C with agitation at 200rpm. The mycelial mat was washed with sterile water and then homogenized. Lysates were clarified by centrifugation and passed through 0.22 ⁇ sterile filters. Protein content was measured.
  • lysates Sterility of lysates was confirmed by the absence of bacterial or fungal growth after incubation for two weeks at 30°C in liquid culture medium. Lysates were stored at -80°C. To use as negative control, water-soluble lysate was prepared from K562 cells using the same homogenization procedure used for the preparation of fungal lysates.
  • PBMC Peripheral blood mononuclear cells
  • PMSC Peripheral blood stem cells
  • G-CSF granulocyte-colony stimulating factor
  • HPC Hematopoietic progenitor cells
  • Platelet-reduced HPCs were incubated with 40 ⁇ g (10 ⁇ g/mL) of each fungal lysate ⁇ A. fumigatus, A, terreus, C. krusei or C. krusei and A. terreus) and ⁇ g/ml CliniMACS® anti-CD28 pure AB reagent in complete AIM-VTM medium supplemented with 10% heat-inactivated auto-plasma for 16-18 hours at 37°C and 5% C0 2 . Unstimulated cells served as a negative control.
  • CD137 Activated antigen specific T cells
  • MS magnetic column Miltenyi Biotec
  • All CD137-enriched fungal T cells comprised at least 50% CD3 + CD137 + cells (63.6 ⁇ 14.4%) from which at least 75% (82.3 ⁇ 6.9%) were CD4 + cells.
  • the CD 137 " cell population was irradiated at 30 Gy and co-cultured with the corresponding CD137-enriched population in complete AIM-VTM medium supplemented with 20 U/mL IL-2, 200 U/mL IL-7 and 200 U/mL IL-15 at 37°C and 5% C0 2 . Cytokines were replenished every 2-3 days and half media exchanges were performed as required. All fungal T cell cultures were maintained for a total of 11 to 12 days (Figure 1A). Individually stimulated T cell cultures generated greater than 96% CD3 + cells (98.6% ⁇ 0.8), of which greater than 85% (92.9% ⁇ 2.8) were CD4 + cells and less than 1 1% (3.2% ⁇ 3.1) were CD8 + cells.
  • T cells The presence of fungus specific T cells was assessed in cultures stimulated with lysates to A. fumigatus, A. terreus, C. krusei or C. Jcrusei and A. terreus by measuring intracellular expression of INFy, TNFa and/or IL-17 following re-stimulation with the same fungal lysate used to originate the T cell culture ( Figures 1B-D, H).
  • Table 1 Secretion of TNFa and IFN- ⁇ cytokines from CD4+ T cells from cultures initially stimulated with A. fumigatus, C krusei or A terreus
  • Table 2 Secretion of cytokines TNFa, IFN- ⁇ , and IL-17 from CD4+ T cells from cultures initially stimulated with C krusei and A terreus
  • T cell cultures were established from platelet-reduced HPCs using synthetic peptide mixes of CMV, EBV, AdV ( ⁇ g/mL per peptide) and a water soluble lysate of A. fumigatus (10 ⁇ g/mL) for antigenic stimulation.
  • CD137 + cells were isolated using immunomagnetic enrichment and culture expanded as described above.
  • T cells producing IFNy in response to antigenic stimulation was also dominated by CMV compared to AdV and A. fumigatus (Figure 2C).
  • the dominance of CMV and EBV in the T cell culture is likely to reflect a higher frequency in the starting population compared to AdV and A. fumigatus specific T cells and/or a competitive advantage during cell expansion in culture.
  • T cell cultures were generated by stimulation with a single antigen (synthetic peptides of CMV, EBV, AdV ( ⁇ g/mL per peptide) or a water soluble lysate of A. fumigatus (4( ⁇ g/mL)) as described above.
  • a single antigen synthetic peptides of CMV, EBV, AdV ( ⁇ g/mL per peptide) or a water soluble lysate of A. fumigatus (4( ⁇ g/mL)) as described above.
  • TNFa production in response to antigenic stimulation The specificity of the individual T cell products was assessed by determining TNFa production in response to antigenic stimulation.
  • the predominant cell type was CD3 + T cells in all cultures (mean 98.6 ⁇ 1.4%).
  • the mean percentage of T cells producing TNFa in response to CMV and EBV was 62.7 ⁇ 17.7% and 73 ⁇ 20.1% respectively.
  • the proportion of T cells with AdV specificity was higher (59.3 ⁇ 16.4%) compared to responses when pooled with other pathogens (2.4 ⁇ 3.5%).
  • 38.4 ⁇ 23.1% of T cells produced TNF in response to an A. fumigatus lysate when prepared separately compared to 0.9 ⁇ 1.1% when prepared in a pooled culture (Figure 3B).
  • the proportion of responding cells (% of the highest of TNFa+ cells from CD4 population, INFy+ cells from CD 8 population or CD107a/b+ cells from CD8 population) that were specific for individual viral or fungal antigens was higher in the individual T cell cultures compared to the multipathogen (Figure 3C).
  • WT1 specificity was measured by CD 107 expression after re-stimulation with
  • WT1 peptide mixture WT1 mean 14.6% ⁇ 7.3; control mean 1.5% ⁇ 0.3; Figure 4B).
  • the proportion of IFN- ⁇ CD8 T cells specific for WT1 was also determined (Figure 4C).
  • T cell products prepared from single T cell pathogen products pooled in defined ratios were prepared and compared to T cell products prepared from multipathogen T cell cultures.
  • T cell products were prepared using CMV, EBV, AdV or A. fumigatus alone or a combination of CMV, EBV, AdV and A. fumigatus as antigenic stimulation as previously described. Following stimulation cells were CD 137 immunoselected and culture expanded as described above.
  • T cell products were generated with a total of ⁇ 2xl0 7 /m 2 ( ⁇ 4xl0 7 total nucleated cells).
  • Cells from individual CMV, EBV, AdV and A. fumigatus T cell cultures were pooled in equal numbers (i.e., lxl 0 7 cells/pathogen) at the completion of the culture period.
  • CMV CMV (21%) specific responses, with 9% responding to AdV and 3% to Asp.
  • CMV 26%, EBV 28%, AdV 25%, Asp 21%; Figure 5 the proportion of responding T cells was distributed more evenly in respect of each pathogen.
  • donor 2 89% of responding T cells in the multipathogen T cell culture were specific for CMV, whereas when single pathogen cultures were combined with equal numbers, the proportion of responding T cells specific for CMV, EBV, AdV and Asp was more evenly distributed (CMV 32%, EBV 36%, AdV 25%, Asp 7%); Figure 5). Based on clinical situation
  • a T cell product is prepared, using methods described above, based on the likelihood of severe infection associated with demographics and the type of transplant and is constituted based on combining equal or varying numbers of cells from the individual pathogen and/or malignant directed cell cultures based on a specific number of:
  • CD3 + cells or CD4 + cells or CD8 + cells from individual cell cultures or
  • a T cell product is prepared, using methods described above, based on the underlying malignancy diagnosis.
  • a defined composition is prepared containing a varying (1-99%) proportion of cells from a culture known to contain T cells specific for the malignant cells relevant to the patient's diagnosis (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma or lymphoma) in combination with one or more infectious pathogen directed cells.
  • a patient receiving a blood stem cell transplant for acute myeloid leukemia is selected for treatment with T cell compositions reactive to both tumour and viral antigens.
  • a T cell product is prepared from single T cell cultures specific to viral and/or fungal antigens CMV, EBV, AdV and VZV and multiple fungi and the tumour antigen WT1, as described above. Following transplant, the patient receives the T cell product to treat and prevent infection.
  • a defined T cell product is prepared, using methods described above, based on the serology or infectious status of the patient and/or donor.
  • a T cell product is prepared by varying the number of T cells based on the presence or likely presence of that pathogen.
  • T cells with specificity to those one or more pathogens may also be included in the composition. Based on the patient's previous infectious history
  • a defined T cell product is prepared, using the methods described above, based on a patient's history of infections. Cultures are established by exposure to pathogens to which the patient is known to have suffered an infection pre-transplant (e.g., a fungal infection or a viral infection, such as CMV, EBV, HSV, VZV, RSV, influenza or tuberculosis).
  • an infection pre-transplant e.g., a fungal infection or a viral infection, such as CMV, EBV, HSV, VZV, RSV, influenza or tuberculosis.
  • a defined T cell product is prepared, using the methods described above, based on a known profile of infection taking into consideration demographic parameters such as age and transplant type.
  • a composition for a paediatric transplantation patient is prepared using the methods described above and comprises T cells reactive to AdV.
  • a composition for a cord transplant recipient is prepared using methods described above and comprises T cells reactive to HHV6.
  • Example 6 Generation and further characterisation of fungus-specific T cell cultures
  • T cell cultures were established from platelet-reduced HPCs and water soluble lysate of A. terreus and C. krusei for antigenic stimulation.
  • CD137 + cells were isolated using immunomagnetic enrichment and culture expanded as described above. Cells were stained with monoclonal antibodies to antigens of interest and analysed by flow cytometry.
  • Cytokine expression was also analysed in T cells cultured for 5 hours with non- pulsed DCs or fungus-pulsed DCs.
  • An increase in the proportion of CD4+ cells expressing interferon-gamma, GM-CSF, IL-2, IL-4, TNF-a, IL-13 and IL-17 was observed following fungal antigen presentation by DCs to fungus-specific T cells (Figure 9). Together, these results show that fungus-specific T cells mediate a proinflammatory / antifungal response when exposed to fungi.
  • T cells that had been cultured for 5 hours with non-pulsed DCs or fungus lysate pulsed DCs were stained with a panel of monoclonal antibodies labelled with heavy metal ion tags and analysed by time-of-flight mass spectrometry, t-distribution stochastic neighborhood embedding (t-SNE) analyses indicated which cells in the landscape of antigen expression were producing GM-CSF, TNF-a and IFN- ⁇ upon exposure to the fungal antigens.
  • the highest expression of the three cytokines was observed in two subpopulations of CD4+ cells ( Figure 10). Responding fungus- specific T cells correspond to the cell cluster showing higher GM-CSF, IFN and TNF expression, indicative of a majority cells with similar phenotype.
  • An XTT assay was performed to assess the anti-hyphal activity of cultured fungus-specific T cells stimulated with A. terreus and C. krusei. Conidia from A. fumigatus was incubated for 2 hours with fungus-specific T cells alone, peripheral blood white blood cells (WBC) that had not previously been cultured with fungi, and a combination of the two.
  • WBC peripheral blood white blood cells
  • T cell cultures were exposed to A. terreus and C. krusei lysate, enriched for
  • the fungus-specific T cells were incubated with DCs pulsed with fungal lysate and antibodies to various MHC proteins. After a period of incubation, CD4 + fungus-specific T cells were analysed for expression of TNF-a. Recognition of fungal antigens by CD4 + fungus-specific T cells was largely mediated by HLA DR ( Figure 12A). CD4 + fungus-specific T cells could recognise fungal antigens even when only one of the two HLA DR antigens was matched between the DC and CD4 + fungus-specific T cells ( Figure 12B).
  • CD4 + fungus-specific T cells generated using cells from the most common HLA types could still recognise fungal antigens presented by HLA DR subtypes that were allelic mismatches (HLA DRBl 01:02; 04:03, 04:04, 04:05; 11:04; 13:02; 15:02) indicating that a bank of fungus-specific T cells made using the most common HLA DR types may be valuable in treating patients with less common HLA DR allelic subtypes who have invasive fungal infection ( Figure 12C-G).
  • Intracellular flow cytometry was conducted to assess TNF-a production in fungus-specific T cells stimulated with HLA DR homozygous B cell lymphoblastoid cell lines (B-LCL).
  • B-LCL effectively presented fungal antigens to CD4 + T cells, highlighting a novel method for determining the HLA DR element of restriction of fungus-specific T cells ( Figure 13). Stimulation can be carried out with HLA DR homozygous B-LCL expressing one of the HLA DR molecules of the fungus-reactive cells.
  • a fungus-reactive product is generated from a donor who is HLA DR 01 :01 and 07:01
  • the product may be stimulated with B-LCL homozygous for HLA DR 01 :01 and with B-LCL homozygous for HLA DR 07:01 to determine which one of the HLA DRs is restricting the activity (or to determine the relative contribution of each HLA DR).
  • This approach may be used for other CD4 + dominated compositions, eg, adenovirus- and HHV6-specific T cell compositions.
  • T cell cultures were established from platelet-reduced HPCs and water soluble lysate of Aspergillus fumigatus for antigenic stimulation.
  • CD137 + cells were isolated using immunomagnetic enrichment and culture expanded as described above.
  • Fungus-specific T cell products were subjected to PCR amplification of TCR- ⁇ , followed by sequencing and read frequency determination.
  • a relatively small number of TCR clones made up the top 25% of TCR sequences ( Figure 14).
  • Lymphoma was diagnosed at age 22 with bulky mediastinal mass, pericardial involvement with tamponade and SVC obstruction as well as disease in multiple areas above and below the diaphragm. The patient had been treated with 6 cycles of R-CHOP chemotherapy and 4 doses of intrathecal MTX with complete response. Lymphoma has remained in remission.
  • a diagnosis of trisomy 21 was made within 2 years following chemotherapy for lymphoma.
  • the patient was treated with arabinoside and idarubicin chemotherapy but experienced complications of neutropenic infection and perianal infection.
  • the EBV and Aspergillus cell product was prepared using two separate cell cultures. 5 x 10 donor mononuclear cells were stimulated with an EBV consensus peptide pool and an Aspergillus fumigatus water soluble lysate. After 11 days of culture, EBV and Aspergillus cultures were enumerated and pooled to create a single multi-pathogen composition comprising 2 x 10 /m EBV-specific T cells and 2 x 10 7 /m 2 Aspergillus-specific T cells (57% CD8, 42% CD4 with 86% of CD8 specific for EBV and 30.9% of CD4 specific for Aspergillus).
  • the WTl cell product was prepared by stimulating 1 10 mononuclear cells with a WTl overlapping peptide mix. Following an 18 hour incubation, activated antigen-specific T cells were isolated by magnetic selection of CD137-expressing T cells and expanded separately by co-culture with irradiated autologous feeder cells in media supplemented with IL-2, IL-7 and IL-15. WTl -specific T cells were cultured for 20 days and cryopreserved in four separate bags containing 4 x 10 7 /m 2 WTl-specfic T cells.
  • EBV- and Aspergillus-specific T cells comprising EBV- and Aspergillus-specific T cells at a dose of 1 x 10 /m EBV-specific T cells and 1 x 10 7 /m 2 Aspergillus-specific T cells.
  • the patient also received on day 35 post-transplant an infusion of the composition comprising WT1 -specific T cells.
  • An infusion of 2 x 10 7 /m 2 WT1 -specific T cells was performed on day 35 post-transplant with 3 additional infusions of the same dose being delivered 4 weeks apart (WT1- specific T cells were administered every four weeks after the first infusion). No infusion-related toxicities and no infectious complications were identified post- infusion. No graft versus host disease was detected.
  • the patient remains well as an outpatient in morphological remission of her AML 4.5 months after transplant.
  • Example 8 Treatment of disseminated fungal infection with fungus-specific T cells
  • a 51 year old male was diagnosed with Hodgkin's lymphoma at age 27 and was treated with 6 cycles of adriamycin, bleomycin, vinblastine, dacarbazine (ABVD) / nitrogen mustard, vincristine, procarbazine, prednisone (MOPP) chemotherapy and radiation to the mediastinum.
  • Hodgkin's disease was cured, but twenty years later the patient developed bilateral pleural effusions of unclear aetiology requiring pleurodesis. The patient suffered from chronic hepatitis B and received long term entecavir.
  • the patient was diagnosed with Waldenstrom's macroglobulinaemia in 2006 after finding elevated levels of IgM.
  • the patient was initially treated with chlorambucil and prednisone (4 cycles).
  • the patient was administered rituximab plus cyclophosphamide, vincristine and prednisone (4 cycles + 2 further cycles of rituximab).
  • the patient was treated for immune-mediated thrombocytopenia with danazol.
  • the patient was treated with rituximab plus bendamustine and idelalisib (2 cycles), but treatment was complicated by severe shingles, and hemolysis related to bendamustine. Bendamustine was ceased and rituximab was continued plus idelalisib maintenance.
  • the patient developed neck swelling due to Scedosporium boydii infection in neck soft tissue.
  • the patient was treated for three months with voriconazole, idelalisib was ceased for 10 months and recommenced in October 2015 in the setting of rising IgM.
  • Aspergillus fumigatus lysate-stimulated T cells were generated from the patient's fully matched HLA, A, B, C, DR Bl healthy identical male sibling. The inventors had previously demonstrated that Aspergillus fumigatus lysate-stimulated T cells are reactive against Scedosporium.
  • Cells were produced by overnight incubation of donor mononuclear cells with Aspergillus fumigatus lysate, selection of CD137 expressing cells using antibody and magnetic bead isolation, and 12 day culture in medium supplemented with cytokines IL-2, IL-7 and IL-15. Following completion of the culture and quality testing, the cells were cryopreserved according to standard laboratory practice. Cells at the end of the culture had the following characteristics: CD3+ 97%, CD4+ 91.1%, CD8+ 7.8%, NK cells 0.4%, monocytes 0%, B cells 0%. Upon stimulation with Aspergillus lysate in vitro, cultured cells had the following characteristics by surface (CD 107) and intracellular (interferon- ⁇ , tumour necrosis factor-a) flow cytometry:
  • CD3+ 7.7% CD107+, 24.6% interferon-y+, 38.7% tumour necrosis factor-a+
  • CD8+ 7.3% CD107+, 11.3% interferon-y+, 15.4% tumour necrosis factor-a+;
  • CD4+ 11.5% CD107+, 26.3% interferon-Y+, 38.7% tumour necrosis factor-a+.
  • Ixl0 6 /m 2 Aspergillus-specific T cells were infused to the patient without prior conditioning.
  • Peripheral blood mononuclear cells were taken pre-infusion and at various times after infusion from day +1 to day +28 post-infusion.
  • Cells were stained with antibodies complexed to heavy metal ions prior to analysis by mass cytometry time of flight (mass CyTOF). Cells were briefly incubated with monensin prior to staining, but were not restimulated in vitro. No acute infusion related toxicities were observed.
  • T cells are cryopreserved for adoptive T cell transfer.
  • a freezing mix containing 40%> saline, 40% Albumex20 and 20% DMSO is prepared.
  • the saline is added to the DMSO and chilled before adding the Albumex20.
  • the freezing mix is kept chilled until required.
  • the cells for cryopreservation are resuspended, pooled and mixed thoroughly.
  • the cells are counted using a haemocytometer and the cell concentration and total cell viability is determined.
  • the cells are spun at 1400rpm for 5 mins and 5mls of the supernatant is removed for mycoplasma testing. The remaining supernatant is discarded. The cells are washed with up to 50ml of 0.9% saline supplemented with Albumex20 and spun at 1400rpm for 5 mins.
  • the cells are resuspended in 0.9% saline at a concentration of 2x10 7 cells/mL.
  • the maximum volume of cells to be added per bad is to be calculated using the formula:
  • the number of bags and quality assurance samples to be cryopreserved is determined, noting that three pilot vials containing 200 ⁇ 1 of T lymphocyte suspension must be stored for post thaw analysis and endotoxin testing; 200 ⁇ 1 of T lymphocyte suspension is to be submitted for mycoplasma testing; three lmL aliquots of T lymphocyte suspension must be stored for quality control testing of the product; and any excess that does not equal a complete dose can be stored in additional aliquots.
  • An equal volume of freezing mix is added to the T lymphocyte suspension and mixed.
  • the required volume of cells is transferred into cryopreservation bags and/or vials.
  • the bags and vials are immediately placed into pre-cooled rate controlled freezers to begin cryopreservation.
  • the T cell products are used in adoptive transfer provided they meet the following release criteria:
  • B cells CD19 +
  • monocytes CD14 +

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Abstract

La présente invention concerne des compositions comprenant des lymphocytes T isolés, présentant une activité contre un antigène fongique, un antigène viral ou un antigène tumoral, la composition comprenant un nombre défini ou un rapport défini de lymphocytes T. L'invention concerne également des compositions comprenant au moins deux populations de lymphocytes T, les compositions étant appropriées pour traiter diverses maladies et divers troubles.
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US11963979B2 (en) 2011-12-12 2024-04-23 Allovir, Inc. Process for T cell expansion
US11981923B2 (en) 2012-02-09 2024-05-14 Baylor College Of Medicine Pepmixes to generate multiviral CTLS with broad specificity
US11931408B2 (en) 2015-09-18 2024-03-19 Baylor College Of Medicine Immunogenic antigen identification from a pathogen and correlation to clinical efficacy
US11951127B2 (en) 2017-06-22 2024-04-09 The Westmead Institute for Medical Research Adoptive T cell therapy 2
WO2021021937A1 (fr) * 2019-07-29 2021-02-04 Baylor College Of Medicine Banques de lymphocytes t spécifiques de l'antigène et leurs procédés de fabrication et d'utilisation thérapeutique

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US11951127B2 (en) 2024-04-09
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US20220226373A1 (en) 2022-07-21
AU2021286255A1 (en) 2022-01-06

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